Virtually every area of biomedical sciences is in need of a system to assay chemical and biochemical reactions and determine the presence and quantity of particular analytes. This need ranges from the basic science research lab, where biochemical pathways are being mapped out and their functions correlated to disease processes, to clinical diagnostics, where patients are routinely monitored for levels of clinically relevant analytes. Other areas include pharmaceutical research, military applications, veterinary, food, and environmental applications. In all of these cases, the presence and quantity of a specific analyte or group of analytes, needs to be determined.
For analysis in the fields of chemistry, biochemistry, biotechnology, molecular biology and numerous others, it is often useful to detect the presence of one or more molecular structures and measure binding between structures. The molecular structures of interest typically include, but are not limited to, cells, antibodies, antigens, metabolites, proteins, drugs, small molecules, proteins, enzymes, nucleic acids, and other ligands and analytes. In medicine, for example, it is very useful to determine the existence of a cellular constituents such as receptors or cytokines, or antibodies and antigens which serve as markers for various disease processes, which exists naturally in physiological fluids or which has been introduced into the system. Additionally, DNA and RNA analysis is very useful in diagnostics, genetic testing and research, agriculture, and pharmaceutical development. Because of the rapidly advancing state of molecular cell biology and understanding of normal and diseased systems, there exists an increasing need for methods of detection, which do not require labels such as fluorophores or radioisotopes, are quantitative and qualitative, specific to the molecule of interest, highly sensitive and relatively simple to implement.
Numerous methodologies have been developed over the years to meet the demands of these fields, such as Enzyme-Linked Immunosorbent Assays (ELISA), Radio-Immunoassays (RIA), numerous fluorescence assays, mass spectroscopy, colorimetric assays, gel electrophoresis, as well as a host of more specialized assays. Most of these assay techniques require specialized preparations, especially attaching a label or greatly purifying and amplifying the sample to be tested. To detect a binding event between a ligand and an antiligand, a detectable signal is required which relates to the existence or extension of binding. Usually the signal is provided by a label that is conjugated to either the ligand or antiligand of interest. Physical or chemical effects which produce detectable signals, and for which suitable labels exist, include radioactivity, fluorescence, chemiluminescence, phosphorescence and enzymatic activity to name a few. The label can then be detected by spectrophotometric, radiometric, or optical tracking methods. Unfortunately, in many cases it is difficult or even impossible to label one or all of the molecules needed for a particular assay. Also, the presence of a label may make the molecular recognition between two molecules not function for many reasons including steric effects. In addition, none of these labeling approaches determines the exact nature of the binding event, so for example active site binding to a receptor is indistinguishable from non-active-site binding such as allosteric binding, and thus no functional information is obtained via the present detection methodologies. Therefore, a method to detect binding events that both eliminates the need for the label as well as yields functional information would greatly improve upon the above mentioned approaches.
Other approaches for studying biochemical systems have used various types of dielectric measurements to characterize certain classes of biological systems such as tissue samples and cellular systems. In the 1950""s, experiments were conducted to measure the dielectric properties of biological tissues using standard techniques for the measurement of dielectric properties of materials known at the time. Since then various approaches to carrying out these measurements have included frequency domain measurements, and time domain techniques such as Time Domain Dielectric Spectroscopy. In these approaches, the experiments were commonly carried out using various types of coaxial transmission lines, or other transmission lines and structures of typical use in dielectric characterization of materials. This included studies to look at the use and relevance of the dielectric properties of a broad range of biological systems: The interest has ranged from whole tissue samples taken from various organs of mammalian species, to cellular and sub-cellular systems including cell membrane and organelle effects. Most recently, there have been attempts to miniaturize the above-mentioned techniques (see e.g., U.S. Pat. Nos. 5,653,939; 5,627,322 and 5,846,708) for improved detection of changes in the dielectric properties of molecular systems. Typically these use the biological samplexe2x80x94be it tissues, cellular systems, or molecular systems-as a shunt or series element in the electrical circuit topology. This configuration has several drawbacks, including some substantial limitations on the frequencies useable in the detection strategy, and a profound limitation on the sensitivity of detecting molecular systems.
In general, limitations exist in the areas of specificity and sensitivity of most assay systems. Cellular debris and non-specific binding often cause the assay to be noisy, and make it difficult or impossible to extract useful information. As mentioned above, some systems are too complicated to allow the attachment of labels to all analytes of interest, or to allow an accurate optical measurement to be performed. Further, a mentioned above, most of these detection technologies yield no information on the functional nature of the binding event. Therefore, a practical and economical universal enabling which can directly monitor without a label, in real time, the presence of analytes or the extent, function and type of binding events that are actually taking place in a given system would represent a significant breakthrough.
More specifically, the biomedical industry needs an improved general platform technology which has very broad applicability to a variety of water-based or other fluid-based physiological systems, such as nucleic acid binding, protein-protein interactions, small molecule binding, as well as other compounds of interest. Ideally, the assay should not require highly specific probes, such as specific antibodies and exactly complementary nucleic acid probes; it should be able to work in native environments such as whole blood, cytosolic mixtures, as well as other naturally occurring systems; it should operate by measuring the native properties of the molecules, and not require additional labels or tracers to actually monitor the binding event; for some uses it should be able to provide certain desired information on the nature of the binding event, such as whether or not a given compound acts as an agonist or an antagonist on a particular drug receptor, and not function simply as a marker to indicate whether or not the binding event has taken place. For many applications, it should be highly miniaturizable and highly parallel, so that complex biochemical pathways can be mapped out, or extremely small and numerous quantities of combinatorial compounds can be used in drug screening protocols. In many applications, it should further be able to monitor in real time a complex series of reactions, so that accurate kinetics and affinity information can be obtained almost immediately. Perhaps most importantly, for most commercial applications it should be inexpensive and easy to use, with few sample preparation steps, affordable electronics and disposable components, such as surface chips for bioassays that can be used for an assay and then thrown away, and be highly adaptable to a wide range of assay applications.
It is important to note that other industries have similar requirements for detection, identification or additional analysis. While most applications involve the use of biological molecules, virtually any molecule can be detected if a specific binding partner is available or if the molecule itself can attach to the surface as described below.
The present invention fulfills many of the needs discussed above and other needs as well.
The present invention provides systems and methods for detecting molecular binding events and other environmental effects using the unique dielectric properties of the bound molecular structure or structures, and the local environment, and also identifying the presence and concentrations of molecular species, as well as physical properties of the local environment, in a particular biological system.
In a first embodiment of the invention, a method for detecting a molecular binding event includes the steps of providing a signal path and a molecular binding layer, which is formed along the signal path. A test signal is propagated along the signal path and couples to the molecular binding layer. In response to the coupling, the signal exhibits a response which is indicative of both the molecular binding event and the molecular binding layer itself.
In a second embodiment of the invention, a method for determining the classification of an unknown ligand is presented. The method comprises the steps of providing a signal path coupled to a first molecular binding layer having N respective antiligands for binding to N respective ligand sub-structures. Next a solution containing a number of unknown ligands is applied to the said molecular binding layer. In response, a second molecular binding layer is formed along the signal path, the second molecular binding layer having N ligands. N respective test signals are propagated to the N respective ligands. N known signal responses defining a known ligand classification are provided. Finally, each of the test signals couples to the N ligand/antiligand complexes, and in response exhibits N respective measured responses indicative of the presence of each of said N sub-structures, so that if a predetermined number of said N known signal responses correlates within a predefined range with the N measured responses, the ligand is determined to be within the known classification.
In a third embodiment of the invention, a method for identifying an unknown molecular binding event is presented. The method includes the steps of providing a signal path, applying a first solution containing a first ligand over the signal path, and forming, in response, a first molecular binding layer along the signal path, whereby the first molecular layer includes the first ligand and is positioned along the signal path and the first solution. A first test signal is propagated along the signal path, the portion of which includes the molecular binding layer comprises a continuous transmission line, whereby the signal couples to the molecular binding layer and in response exhibits a first signal response. A known signal response corresponding to a known molecular binding event is provided and the first signal response is then compared to the known signal response, wherein if the first signal response correlates to the known signal response within a predefined range, the unknown molecular binding event comprises the known molecular binding event.
In a fourth embodiment of the invention, a method for quantitating an unknown concentration of ligands in solution is presented. The method includes the steps of providing a signal path which is coupled to a first molecular binding layer having at least one antiligand, applying a solution having a known concentration of ligands over the molecular binding layer, and propagating a test signal along the signal path. Next a first signal response is measured and an extrapolation algorithm is generated. A second test signal is subsequently propagated and a second signal response is measured. The second signal response is then correlated to the algorithm.
In a fifth embodiment of the invention, a bio-electrical interface is provided for detecting the presence of a ligand in a solution. The bio-electrical interface includes a signal path, a solution for providing the ligand and a molecular binding layer. The molecular binding layer includes the ligand and is coupled along the signal path and the solution.
In a sixth embodiment of the invention, a bio-assay device is provided for detecting one or more properties associated with a molecular binding layer, such as the presence of a ligand, using a test signal. The apparatus includes a signal path having a first port and a second port for communicating the test signal, and a continuous conductive region therebetween. The bio-assay device further includes a molecular binding layer, which may have a ligand, and which is coupled to the signal path. The bio-assay device may further include a solution coupled to said molecular binding layer, which may transport the ligand to the molecular binding layer.
In a seventh embodiment of the invention, a system for detecting a molecular binding event is presented. The system includes a signal source for launching a test signal, a bio-assay device coupled to said signal source and a second detector coupled to the bio-assay device. The bio-assay device includes a signal path and a first molecular binding layer, which may include a ligand or antiligand, and which may be coupled to a solution and the signal path. The test signal propagates along the signal path, which is continuous throughout the region of the molecular binding layer, and couples to the molecular binding layer, and in response exhibits a signal response which indicates the presence of said molecular binding event.
In one aspect, the present invention is the use of the interaction of electromagnetic radiation, typically between about 1 MHz and 1000 GHz, with molecular structures in a molecular binding layer to determine properties of the structures, such as dielectric properties, structural properties, binding events and the like. Also, the present invention uses a test signal on a bio-electrical interface having a signal path along which the molecular binding layer is coupled to detect analytes therein.
The nature and advantages of the present invention will be better understood with reference to the following drawings and detailed description.